The signaling pathways by which NO protects against stroke injury are mediated by soluble guanylyl cyclase activation and cyclic guanosine monophosphate ( cGMP) formation. However, the mechanisms of downstream signaling from cGMP to smooth muscle cells (SMC) remain incompletely understood. The cGMP- dependent protein kinase I (cGKI) is a key mediator of cGMP signaling. Our preliminary results indicate that reperfusion of middle cerebral artery was attenuated and stroke injury was more pronounced in cGKI SMC specific inducible conditional knockout mice than in wild-type mice, suggesting a protective role for cGKI in SMC against cerebral reperfusion injury. The overall goal of this project is to identify the molecular mechanisms underlying the protective effect of cGKI signaling in SMC against stroke injury. We will test the hypotheses that cGKI mediate the protective effects of cGMP and that the protective effects of cGMP-cGKI signaling are SMC specific. We will take advantage of mice with inducible knockout of cGKI in SMC to specifically study the contributions of SMC-dependent vascular mechanisms to the protective effects of cGMP against stroke. Our goals are (1) to determine the role of cGKI SMC-dependent mechanisms to the protective effect in stroke and (2) to identify the molecular mechanisms underlying the protective role of cGKI-SMC signaling in stroke injury. These results will make possible the development of new specific therapeutic approaches to stroke. Hypothesis?cGKI in smooth muscle cells protects against stroke. Aim 1: To determine the relevance and mechanism of cGKI-mediated signaling in SMC for stroke protection. We will study whether deficiency of cGKI in SMC worsens stroke outcome in cGKI SMC- specific knockout mice. We will compare stroke outcome in cGKI knockout mice and cGKI wild-type mice. We will determine neurological deficit and infarct volume after occlusion and reperfusion of the middle cerebral artery and compare cerebral blood flow at baseline and during ischemia and reperfusion. Aim 2: To study the role of cGKI of SMC in vascular reactivity. We will test whether cGKI deletion in SMC impairs reactivity of isolated vessels, cerebral blood flow and autoregulation. Aim 3: To investigate the role of platelets and leukocytes in stroke injury of cGKI SMC deficient mice. Platelets aggregation and accumulation of leukocytes within cerebral microvessels is critically involved in stroke injury. We will study how cGKI deficiency in SMC affects the role of platelet aggregation and leukocytes accumulation in stroke injury. We anticipate that studies described in this proposal will establish a new mouse model for stroke studies and further our knowledge on the role of cGKI SMC signaling in the protection against stroke. These results will be critical to provide the scientific foundation for the development of cGKI-elevating compounds as therapeutic agents in stroke patients.